Platelet storage methods and compositions for same

ABSTRACT

Disclosed are compositions and methods for slowing, preventing, or reversing platelet damage, particularly as may occur during blood banking or during refrigeration of platelets. The composition may include one or more of a RAC inhibitor, a CDC42 inhibitor, a RHOA inhibitor, or a combination thereof. The compositions may further include a pharmaceutically acceptable carrier.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. application Ser.No. 14/743,213, filed Jun. 18, 2015, of the same title, which claimspriority to and benefit of U.S. Provisional Application Ser. No.62/013,662, filed Jun. 18, 2014, of same title, in their entirety forall purposes.

BACKGROUND

Patients with low platelet counts often require platelet transfusion.This is particularly crucial in the treatment of patients with cancer ormassive trauma. The use of platelet transfusions has increaseddramatically since 1980s, but a safe, long-term platelet storage methodremains unavailable. The demonstration of successful, refrigeratedstorage of platelets for extended lengths of time, for example, 7 daysor longer, would dramatically change the current practice of platelettransfusion in the Western World. Approximately 3,000,000 doses ofplatelets are used in the United States every year, and account forsales of ˜$1.5 Billion annually. The current short shelf-life representsa major handicap to convert platelet products into effectivecommodities. Depending on the time of the year, month or even week, upto 20% of products can be wasted due to expiration. In the meantime,there are moments of platelets shortages due to unpredictable increasedusage. The extension of platelet product shelf-life would strengthen thenational inventory of platelets for oncological and trauma patients. Anestimated 10-fold increase in the need of platelet and plasma productsis expected by the US government in war casualties and massive traumapatients due to the 1 red cell: 1 platelet: 1 plasma product transfusionpolicy.

Current practice has platelets stored at 20 to 24° C. after preparation,which has a limited lifetime up to 5 days, primarily due to concernsabout bacterial contamination. Bacterial contamination of plateletproducts for transfusion is a major safety problem in blood banking. Theconsequence of transfusion of contaminated products is increasedmorbi-mortality among a susceptible population of cancer patients (1).Different technologies have been developed aiming to minimize the riskof bacterial contamination including diversion pouches for collection,bacterial detection with automatic culture systems and pathogenreduction systems (2-6). While there has been a significant reduction inthe number of cases of platelet transfusion associated sepsis, the riskof transfusion-associated sepsis ranges between 1 in 15,000 to 86,000platelet transfusions (7, 8). Storage of platelets in cold temperatures,as is done for red cells, would reduce the proliferation of mostbacteria and allow a longer period of storage (9), minimizing thecurrent shortages (10) that the short storage time (5-day) for plateletsapproved by the FDA (11). Conventional cold storage of platelets,however, has been hampered by the discovery that the 24-hour recovery ofchilled platelets was significantly reduced (14).

The development of a method to prevent platelet damage uponrefrigeration is a much needed, and long sought after advance in bloodbanking. Such development would revolutionize the current method ofplatelet storage. The instant disclosure solves one or more of thesedeficiencies in the art.

BRIEF SUMMARY

Disclosed are compositions and methods for one or more of slowing,preventing, or reversing platelet damage, particularly as may occurduring blood banking or during refrigeration of platelets. Thecomposition may include one or more of a RAC inhibitor, a CDC42inhibitor, a RHOA inhibitor, or a combination thereof. The compositionsmay further include a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict a schematic model of “cold receptor” initiatedintracellular events involving Rho GTPases. FIG. 1A. Cold causes theactomyosin changes, Ca2+ mobilization, loss of spectrin anchorage, andGplb clustering. FIG. 1B. “Cold receptor” may stimulate Cdc42, Rac,and/or Rho activation, which in turn control lipid raft assembly andactomyesin reorganization, resulting in Gplb clustering.

FIG. 2 depicts Rho GTPases inhibitor chemical structures and targets.

FIGS. 3A-3D depict a docking model of Casin bound to Cdc42 surfacegroove and data demonstrating Casin activity. FIG. 3A. Docking model ofCasin bound to a Cdc42 surface groove required for activation. FIG. 3B.Collagen-induced Cdc42 activation is inhibited by Casin (10 μM) in aGST-PAK pulldown assay. FIG. 3C. Fibrinogen mediated platelet actinfilopedia structure is blocked by Casin (10 μM). Rhodamine conjugatedphalloidin-staining of platelets adhered to coated collagen surface.FIG. 3D. Collagen induced aggregation is blocked by 10 μM Casin (upperpanel) and is reversible (up to 30 μM Casin) upon a wash of theinhibitor treated platelets (lower panel).

FIGS. 4A-4E depict a docking model of Rac inhibitor NSC23766 and datashowing NSC23766 activity. FIG. 4A. X-ray structure of NSC23766 bound toa Rac1 surface groove required for GEF activation. FIG. 4B. Rac1activity is inhibited by NSC23766 (50 μM) in a GST-PAK pulldown assay.FIG. 4C. Fibrinogen-mediated platelet actin lamellopodia structure isblocked by NSC23766 (50 μM). Rhodamine conjugated phalloidin-staining ofplatelets adhered to coated collagen surface. FIG. 4D. Collagen inducedaggregation is blocked by NSC23766 in a dose-dependent fashion. FIG. 4E.Inhibition of collagen induced platelet aggregation by NSC23766 (50 μM)is reversible upon a wash of the inhibitor treated platelets.

FIGS. 5A-5D depict a docking model of Rho inhibitor G04 and data showingG04 activity. FIG. 5A. Docking model of G04 bound to a RhoA surfacegroove required for GEF activation. Upper panel: low resolution; Lowerpanel: high resolution binding domain. FIG. 5B. Collagen-induced RhoAactivity is inhibited by Go4 (50 μM) in a GST-Rhotekin pulldown assay.FIG. 5C. U46629 (10 mM/Fibrinogen (3 μM)-mediated platelet actinlamellopodia structure is blocked by G04 (30 μM). Rhodamine conjugatedphalloidin-staining of platelets adhered to coated collagen surface.FIG. 5D. Collagen induced aggregation is inhibited by G04 (upper panel).Similar to collagen, thrombin induced aggregation (data not shown) isblocked by G04 in a dose-dependent fashion (upper panel) and isreversible upon a wash of the inhibitor treated platelets (lower panel).

FIGS. 6A-6D depict the effects of Rho GTPase inhibitor treatment onplatelet transfusion. FIG. 6A. Experimental designs. FIG. 6B-FIG. 6D.24-hour recovery and survival of platelets in different conditions. FIG.6B. 24-hour recovery. FIG. 6C-FIG. 6D. Platelet recovery at differenttime points. Donor platelets were stored at room temperature (squares)or pretreated with a mixed Rho GTPase inhibitors (50 μM NSC23766, 10 μMCASIN and/or 75 μM G04) or no drug (control). Data are presented as meanSD. *p<0.01 (Anova test with Bonferroni correction, between refrigeratedwith no inhibitor and refrigerated with triple inhibitor combination).

FIG. 7 depicts effects of Rho GTPase inhibitor combinations on plateletsurvival upon transfusion. Platelet recovery at different time points isshown. Donor platelets were stored at room room temp (squares) orpretreated with a mixed Rho GPase inhibitors (50 uM NSC23766, 10 uMCASIN and/or 75 uM G04) or no drug (Ctrl). Data are presented asmean±SD. *p<0.01 (Anova test with Bonferroni correction, betweenrefrigerated with no inhibitor and refrigerated with triple inhibitorcombination).

FIG. 8 depicts total microparticle and Gplb+ microparticle counts fromplatelets stored in different conditions. Results are representative ofthree independent experiments with similar results of platelets storedfor 6 days in 50 μM NSC23766, 10 μM CASIN and/or 75 μM G04, or no drug(Vehicle control). The left panel shows microparticles/mcL ofrefrigerated stored platelets for 6 days; the right panel shows Gplb+microparticle counts of refrigerated stored platelets for 6 days.

FIG. 9 depicts amelioration of lipid raft formation by Rho GTPaseinhibitor combinations. Platelets were stored at RT or 4° C. for 3 or 7days in different combinations of inhibitors (50 μM NSC23766, 10 μMCASIN and/or 75 μM G04) or no drug (vehicle control) at RT or 4° C.Platelet lysates were analyzed in the presence (lipid rafts) or absence(whole cell lysate) of Triton-X-100 mediated extraction. Results arerepresentative of two independent experiments with similar results. Lowepanels represent normalized quantification of lipid raft formation.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the embodiments belong. Although any methods andmaterials similar or equivalent to those described herein may also beused in the practice or testing of the embodiments, the preferredmethods and materials are now described. All publications mentionedherein are expressly incorporated by reference in their entireties.

DEFINITIONS

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aplatelet” includes a plurality of such platelets and reference to “thecarrier” includes reference to one or more carriers and equivalentsthereof known to those skilled in the art, and so forth.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Where particular values are described inthe application and claims, unless otherwise stated the term “about”meaning within an acceptable error range for the particular value shouldbe assumed.

An “amide” is a chemical moiety with formula —(R)n—C(O)NHR′ or—(R)n—NHC(O)R′, where R and R′ are independently selected from the groupconsisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) and heteroalicyclic (bonded through a ring carbon), and where nis 0 or 1.

The term “aromatic” refers to an aromatic group which has at least onering having a conjugated pi electron system and includes bothcarbocyclic aryl (e.g., phenyl) and heterocyclic aryl groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups. The term“carbocyclic” refers to a compound which contains one or more covalentlyclosed ring structures, and that the atoms forming the backbone of thering are all carbon atoms. The term thus distinguishes carbocyclic fromheterocyclic rings in which the ring backbone contains at least one atomwhich is different from carbon. The term “heteroaromatic” refers to anaromatic group which contains at least one heterocyclic ring.

As used herein, the term “alkyl” refers to an aliphatic hydrocarbongroup. The alkyl moiety may be a “saturated alkyl” group, which meansthat it does not contain any alkene or alkyne moieties. The alkyl moietymay also be an “unsaturated alkyl” moiety, which means that it containsat least one alkene or alkyne moiety. An “alkene” moiety refers to agroup consisting of at least two carbon atoms and at least onecarbon-carbon double bond, and an “alkyne” moiety refers to a groupconsisting of at least two carbon atoms and at least one carbon-carbontriple bond. The alkyl moiety, whether saturated or unsaturated, may bebranched, straight chain, or cyclic.

The alkyl group may have 1 to 20 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 20” refers to each integer inthe given range; e.g., “1 to 20 carbon atoms” means that the alkyl groupmay consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., upto and including 20 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). The alkyl group may also be a medium size alkyl having 1 to10 carbon atoms. The alkyl group could also be a lower alkyl having 1 to5 carbon atoms. The alkyl group of the compounds of the invention may bedesignated as “C1-C4 alkyl” or similar designations. By way of exampleonly, “C1-C4 alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl.

The alkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is(are) one or more group(s) individually andindependently selected from cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato,isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino,including mono- and di-substituted amino groups, and the protectedderivatives thereof. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and the like. Wherever asubstituent is described as being “optionally substituted” thatsubstitutent may be substituted with one of the above substituents.

The term “ester” refers to a chemical moiety with formula —(R)n—COOR′,where R and R′ are independently selected from the group consisting ofalkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon), and where n is 0 or 1.

The substituent “R” appearing by itself and without a number designationrefers to a substituent selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon).

An “O-carboxy” group refers to a RC(═O)O— group, where R is as definedherein.

A “C-carboxy” group refers to a —C(═O)OR groups where R is as definedherein.

An “acetyl” group refers to a —C(═O)CH3, group.

A “trihalomethanesulfonyl” group refers to a X3CS(═O)2- group where X isa halogen.

A “cyano” group refers to a —CN group.

An “isocyanato” group refers to a —NCO group.

A “thiocyanato” group refers to a —CNS group.

An “isothiocyanato” group refers to a —NCS group.

A “sulfinyl” group refers to a —S(═O)—R group, with R as defined herein.

A “S-sulfonamido” group refers to a —S(═O)2NR, group, with R as definedherein.

A “N-sulfonamido” group refers to a RS(═O)2NH— group with R as definedherein.

A “trihalomethanesulfonamido” group refers to a X3CS(═O)2NR— group withX and R as defined herein.

An “O-carbamyl” group refers to a —OC(═O)—N(R)2, group—with R as definedherein.

An “N-carbamyl” group refers to a ROC(═O)NH— group, with R as definedherein.

An “O-thiocarbamyl” group refers to a —OC(═S)—N(R)2, group with R asdefined herein.

An “N-thiocarbamyl” group refers to an ROC(═S)NH— group, with R asdefined herein.

A “C-amido” group refers to a —C(═O)—N(R)2 group with R as definedherein.

An “N-amido” group refers to a RC(═O)NH— group, with R as definedherein.

The term “perhaloalkyl” refers to an alkyl group where all of thehydrogen atoms are replaced by halogen atoms.

The term “acylalkyl” refers to a RC(═O)R′— group, with R as definedherein, and R′ being a diradical alkylene group. Examples of acylalkyl,without limitation, may include CH3C(═O)CH2-, CH3C(═O)CH2CH2-,CH3CH2C(═O)CH2CH2-, CH3C(═O)CH2CH2CH2-, and the like.

Unless otherwise indicated, when a substituent is deemed to be“optionally substituted,” it is meant that the substitutent is a groupthat may be substituted with one or more group(s) individually andindependently selected from cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato,isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino,including mono- and di-substituted amino groups, and the protectedderivatives thereof.

The terms “protecting group” and “protecting groups” as used hereinrefer to any atom or group of atoms that is added to a molecule in orderto prevent existing groups in the molecule from undergoing unwantedchemical reactions. The protecting group moiety may be chosen in such away, that they are stable to the reaction conditions applied and readilyremoved at a convenient stage using methodology known from the art. Anon-limiting list of protecting groups include benzyl; substitutedbenzyl; alkylcarbonyls (e.g., t-butoxycarbonyl (BOC));arylalkylcarbonyls (e.g., benzyloxycarbonyl, benzoyl); substitutedmethyl ether (e.g. methoxymethyl ether); substituted ethyl ether; asubstituted benzyl ether; tetrahydropyranyl ether; silyl ethers (e.g.,trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,or t-butyldiphenylsilyl); esters (e.g. benzoate ester); carbonates (e.g.methoxymethylcarbonate); sulfonates (e.g. tosylate, mesylate); acyclicketal (e.g. dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane or1,3-dioxolanes); acyclic acetal; cyclic acetal; acyclic hemiacetal;cyclic hemiacetal; and cyclic dithioketals (e.g., 1,3-dithiane or1,3-dithiolane).

As used herein, the term “cycloalkyl” is intended to cover three-,four-, five-, six-, seven-, and eight- or more membered rings comprisingcarbon atoms only. A cycloalkyl can optionally contain one or moreunsaturated bonds situated in such a way, however, that an aromaticpi-electron system does not arise. Some examples of “cycloalkyl” are thecarbocycles cyclopropane, cyclobutane, cyclopentane, cyclopentene,cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene,1,4-cyclohexadiene, cycloheptane, or cycloheptene.

As used herein, “heterocyclyl” means a cyclic ring system comprising atleast one heteroatom in the ring system backbone. The heteroatoms areindependently selected from oxygen, sulfur, and nitrogen. Heterocyclylsmay include multiple fused rings. Heterocyclyls may have any degree ofsaturation provided that at least one ring in the ring system is notaromatic. Heterocyclyls may be substituted or unsubstituted, and areattached to other groups via any available valence, preferably anyavailable carbon or nitrogen. Preferred monocyclic heterocycles are of 5or 6 members. In six membered monocyclic heterocycles, the heteroatom(s)are selected from one up to three of oxygen, sulfur, and nitrogen, andwherein when the heterocycle is five membered, preferably it has one ortwo heteroatoms selected from oxygen, sulfur, and nitrogen.

A heterocyclyl can further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclicthioimides, cyclic carbamates, and the like. Some examples of“heterocyclyls” include, but are not limited to, tetrahydrothiopyran,4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane,1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane, 1,4-oxathiin,1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide,succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine,hydantoin, dihydrouracil, morpholine, trioxane,hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran,pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline,pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3-dioxolane,1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, and1,3-oxathiolane. The attachment point of a heterocycle radical can be atthe position of a nitrogen heteroatom or via a carbon atom of theheterocycle.

The term “aryl” means a carbocyclic aromatic ring or ring system.Moreover, the term “aryl” includes fused ring systems wherein at leasttwo aryl rings, or at least one aryl and at least one C3-8-cycloalkylshare at least one chemical bond. Some examples of “aryl” rings includeoptionally substituted phenyl, naphthalenyl, phenanthrenyl, anthracenyl,tetralinyl, fluorenyl, indenyl, and indanyl. The term “aryl” relates toaromatic, including, for example, benzenoid groups, connected via one ofthe ring-forming carbon atoms, and optionally carrying one or moresubstituents selected from heterocyclyl, heteroaryl, halo, hydroxy,amino, cyano, nitro, alkylamido, acyl, C1-6 alkoxy, C1-6 alkyl, C1-6hydroxyalkyl, C1-6 aminoalkyl, C1-6 alkylamino, alkylsulfenyl,alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl. The arylgroup can be substituted at the para and/or meta positions. In otherembodiments, the aryl group can be substituted at the ortho position.Representative examples of aryl groups include, but are not limited to,phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl,3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl,3-methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl,hydroxymethylphenyl, trifluoromethylphenyl, alkoxyphenyl,4-morpholin-4-yl-phenyl, 4-pyrrolidin-1-ylphenyl, 4-pyrazolylphenyl,4-triazolylphenyl, and 4-(2-oxopyrrolidin-1-yl)phenyl.

As used herein, the term “heteroaryl” means an aromatic radical havingone or more heteroatom(s) (e.g., oxygen, sulfur, or nitrogen) in thering backbone and may include a single ring (e.g., pyridine) or multiplecondensed rings (e.g., quinoline). Heteroaryl groups can carry one ormore substituents, each independently selected from halo, hydroxy,amino, cyano, nitro, cycloalkyl, haloalkyl, aryl, heterocyclyl,mercapto, alkylamido, acyl, C1-6-alkoxy, C1-6-alkyl, C1-6-hydroxyalkyl,C1-6-aminoalkyl, C1-6-alkylamino, alkylsulfenyl, alkylsulfinyl,alkylsulfonyl, sulfamoyl, and trifluoromethyl. Representative examplesof heteroaryl groups include, but are not limited to, optionallysubstituted derivatives of furan, benzofuran, thiophene, benzothiophene,pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole,benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole,benzimidazole, pyrazole, indazole, tetrazole, quionoline, isoquinoline,pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3-oxadiazole,1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole,pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine,cinnoline, phthalazine, quinazoline, and quinoxaline. In someembodiments, the substituents can be halo, hydroxy, cyano, O—C1-6-alkyl,C1-6-alkyl, hydroxy-C1-6-alkyl, and amino-C1-6-alkyl.

The term “platelet” is used here to refer to a blood platelet. Aplatelet can be described as a minisule protoplasmic disk occurring invertebrate blood. Platelets play a role in blood clotting. The plateletmay be derived from any source including a human blood supply, or thepatient's own blood.

As used herein, the term “effective amount” means the amount of one ormore active components that is sufficient to show a desired effect. Thisincludes both therapeutic and prophylactic effects. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

Refrigerated storage is believed to reduce platelet life-span due todecreased temperature that cause glycoprotein-Ib (GPIb) receptors tocluster on specific microdomains of the platelet membrane. Applicant hasfound that recognition of specific glycated/syalylated residues onclustered glycoproteins by macrophage β2 integrins and hepatocyteAshwell-Morell receptors results in platelet phagocytosis by the hostand removal from circulation. Thus, Applicant has identified preventionof glycoprotein clustering as a useful target for chemical intervention.

Platelet glycoproteins are intimately associated with intracellularcytoskeleton. Clustering of platelet glycoproteins depends on theformation of lipid raft in the platelet membrane, which in turn dependson the dynamics of the highly regulated processes of actomyosinassembly/disassembly. Rho family GTPases, including RhoA, Rac1 andCdc42, are a class of GTP-binding enzymes that are central regulators ofF-actin polymerization/depolymerization, and have been shown to controllipid raft formation and composition. Therefore, Applicant postulatesthat changes in Rho GTPase activities may influence platelet membranelipid raft assembly and glycoprotein composition. Reversible targetingof Rho family GTPases by small molecule inhibitors may preventcytoskeleton-dependent refrigeration storage lesions in platelets andresult in increased platelet survival.

The mechanisms of how cold temperatures affect platelet survival are notcompletely understood, though significant information has been collectedin the past decade. The effects of cold temperature on platelets arebelieved to be complex and involve shape change, cytoskeletalreorganization, activation, cell surface protein clustering and changesin the carbohydrate structures of surface glycoproteins (15-18).Refrigeration-induced changes including filopodia or lamellipodia areaccompanied by an increase in the fraction of total cellular actin in apolymeric state (F-actin) (12, 18, 19) and disappearance of a peripheralmicrotubule coil (20). Isolated prevention of microtubule polymerizationusing colchicine has not resulted in shape change prevention uponactivation (21, 22). Prevention of isolated actin dynamics usingcytochalasin B results in reversion of discoid shape (18) but not inimproved platelet survival in baboons (23), suggesting that irreversibleblockade of actin polymerization does not prevent the refrigerationdamage.

Presence of platelet cold receptors has been postulated as anexplanation for both homeostatic and clinical effects when platelets aresubmitted to temperatures below 16° C. Cold temperature is believed toinduce deglycosylation of glycoprotein Ib ectodomain exposingN-acetyl-D-glucosamine residues (17), which sequesters GM1 gangliosidesin lipid rafts. Raft-associated glycoprotein Iba forms clusters uponbinding of 14-3-3z adaptor proteins to its cytoplasmic tail, a processaccompanied with mitochondrial damage and PS exposure (apoptosis-like)(24). The mechanisms of platelet clearance are believed to be associatedwith lipid-raft associated GPIb clustering and prevention of clusteringprevents platelet clearance (15, 25). Intimately associated withintracellular cytoskeleton, GPIb clustering depends on the formation ofmicrodomains (so-called “lipid rafts”) in the platelet membrane which inturn depends on the dynamics of the highly regulated processes ofacto-myosin assembly/disassembly at multiple levels. In summary,refrigeration results in multiple, complex platelet defects that may beclosely associated with cytoskeletal impairments at multiple levels(FIG. 1A).

Actin cytoskeletal rearrangements responsible for lipid rafts and GPIbclustering in lipid rafts depends on the coordinated activities ofCdc42, Rac1 and RhoA GTPases, which control specific downstreameffectors in regulating polymerization and depoymerization of F-actin,actomyosin contraction, tubulin polymerization, and spectrin anchorage.The Rho family GTPases are a class of GTP-binding enzymes that act assignaling switches in spatial/temporal transduction and amplification ofsignals from platelet receptors to the intracellular signaling pathwaysthat drive platelet function. Among the direct Rho GTPase effectors,WASPs, formins and PAKs that control F-actinpolymerization/depolymerization have been shown to be crucial in thecontrol of lipid raft formation and composition and tubularpolymerization of platelets (27) (FIG. 1B). Therefore, changes in RhoGTPase activities may influence platelet membrane microdomain assemblyand glycoprotein composition. Earlier studies using dominant negativemutants of Cdc42 and Rac1 found no effect on prevention of cold-inducedplatelet damage (28), but the limitation of the tools used has preventedinvestigators from manipulating actin/actomyosin dynamics in a specific,reversible fashion.

Without intending to be limited by theory, it is believed thatreversible inhibition of multiple Rho family of GTPases by chemicalinhibitors can significantly improve platelet survival and transfusionfunction after refrigerated storage by interference with actomyosindynamics and membrane microdomains to prevent GPIb clustering.

Compositions and methods useful for platelet survival and/or quality,transfusion, and associated issues are disclosed herein. In one aspect,a composition for platelet storage or treatment is described. Thecomposition may comprise a RAC inhibitor, for example, those describedin U.S. Pat. Nos. 7,517,890 and 7,612,080, a CDC42 inhibitor, forexample, those described in U.S. Pat. No. 8,383,124; a RHOA inhibitor,and combinations thereof.

In one aspect, the RAC inhibitor may comprise a compound having thestructure of Formula I or a pharmaceutically acceptable salt thereof:

wherein

R₁ to R₂ are independently selected from the group consisting of H,—X-Alk, —X-Alk-X′, and —X—Y—X′; wherein

X is —CR₇R₈;

X′ is —CHR₇R₈;

Alk is a C₂-C₁₈ substituted or unsubstituted hydrocarbon chain;

Y is a C₂-C₈ substituted or unsubstituted alkylene chain;

R₆ is H or (C₁-C₄)alkyl; and

R₇ and R₈ are independently selected from the group consisting of H and(C₁-C₄)alkyl;

or a salt thereof;

In one aspect, the RAC inhibitor may comprise the structure of FormulaIa or a pharmaceutically acceptable salt thereof.

wherein:

R₁₀ to R₁₂ are independently selected from the group consisting of H,halo, (C₁-C₄)alkyl, branched (C₃-C₄) alkyl, halo (C₁-C₄) alkyl, (C₁-C₄)alkoxy, NO₂, and NH₂.

In one aspect, the RAC inhibitor may comprise Formula Ib or apharmaceutically acceptable salt thereof.

In one aspect, the CDC42 inhibitor may comprise Formula II or apharmaceutically acceptable salt thereof.

wherein

Y is selected from the group consisting of OR₇, NR₈R₉, and NNR₈R₉;

R₇ is selected from the group consisting of C₁₋₆ alkyl, (CH₂)uC₃₋₇cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy, hydroxy-C₁₋₆ alkyl, phenyl, C₁₋₆alkyl substituted with up to 5 fluoro, and C₁₋₆ alkoxy substituted withup to 5 fluoro, said C1-6 alkyl, (CH2)uC₃₋₇ cycloalkyl, C₂₋₆ alkenyl,C₁₋₆ alkoxy, hydroxy-C₁₋₆ alkyl, phenyl are each optionally substitutedwith one or more substituents each independently selected from the groupconsisting of halo, —CN, —OH, C₁₋₆ alkoxyl, heteroaryl, R₁₉, and OR₂₀;

R₈ and R₉ may each be separately a hydrogen, or separately selected fromthe group consisting of C₁₋₆ alkyl, C₃₋₇ cycloalkyl, and phenyl, saidC₁₋₆ alkyl, C₃₋₇ cycloalkyl, and phenyl, each optionally substitutedwith one or more substituents each independently selected from the groupconsisting of halo, cyano, nitro, hydroxy, C₁₋₆ alkyl,—(CH₂)uC₃₋₇cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy, hydroxy-C₁₋₆ alkyl,R₁₉, OR₂₀, C₁₋₆ alkyl substituted with up to 5 fluoro, and C₁₋₆ alkoxysubstituted with up to 5 fluoro, each optionally substituted with one ormore substituents each independently selected from the group consistingof halo, cyano, nitro, hydroxy, C₁₋₆ alkyl, and C₁₋₆ alkoxy; or R₈ andR₉ are optionally taken together with the nitrogen to which they areattached to form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, ormorpholinyl, each optionally substituted with one or more substituentseach independently selected from the group consisting of halo, cyano,nitro, hydroxy, C₁₋₆ alkyl, (CH₂)uC₃₋₇cycloalkyl, C₂₋₆ alkenyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, phenyl, C₁₋₆ alkyl substituted with up to 5fluoro, and C1-6 alkoxy substituted with up to 5 fluoro; or R₈ and R₂come together to be C₁₋₃ alkyl linking together as a ring;

each u is independently 0, 1, 2, 3, or 4;

R₂ is a hydrogen, or selected from the group consisting of C₁₋₆ alkyl,C₃₋₇ cycloalkyl, and phenyl, said C₁₋₆ alkyl, C₃₋₇ cycloalkyl, andphenyl, each optionally substituted with one or more substituents eachindependently selected from the group consisting of halo, cyano, nitro,hydroxy, C₁₋₆ alkyl, —(CH₂)uC₃₋₇ cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy,hydroxy-C₁₋₆ alkyl, phenyl, C₁₋₆ alkyl substituted with up to 5 fluoro,C₁₋₆ alkoxy substituted with up to 5 fluoro, and —O(CH₂)uphenyloptionally substituted with one or more substituents each independentlyselected from the group consisting of halo, cyano, nitro, hydroxy, C₁₋₆alkyl, and C₁₋₆ alkoxy; or R₈ and R₂ come together to be C₁₋₃ alkyllinking together as a ring;

R₃, R₄, R₅ and R₆ are each independently selected from the groupconsisting of: hydrogen, halo, cyano, nitro, hydroxy, C₁₋₆ alkyl,(CH₂)uC₃₋₇cycloalkyl, —O(CH₂)uC₃₋₇cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy,hydroxy-C₁₋₆ alkyl, phenyl, C₁₋₆ alkyl substituted with up to 5 fluoro,and C₁₋₆ alkoxy substituted with up to 5 fluoro, said C₁₋₆ alkyl,(CH₂)uC₃₋₇cycloalkyl, —O(CH₂)uC₃₋₇cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy,hydroxy-C₁₋₆ alkyl, and phenyl, each optionally substituted with one ormore substituents each independently selected from the group consistingof halo, cyano, nitro, hydroxy, C₁₋₆ alkyl, —(CH₂)uC₃₋₇cycloalkyl, C₂₋₆alkenyl, C₁₋₆ alkoxy, hydroxy-C₁₋₆ alkyl, phenyl, C₁₋₆ alkyl substitutedwith up to 5 fluoro, and C₁₋₆ alkoxy substituted with up to 5 fluoro,said phenyl optionally substituted with one or more substituents eachindependently selected from the group consisting of halo, cyano, nitro,hydroxy, C₁₋₆ alkyl, —(CH₂)uC₃₋₇cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy,hydroxy-C₁₋₆ alkyl, phenyl, C₁₋₆ alkyl substituted with up to 5 fluoro,and C₁₋₆ alkoxy substituted with up to 5 fluoro;

R₁₉ is aryl optionally substituted with one or more substituents eachindependently selected from the group consisting of halo, cyano, nitro,hydroxy, C₁₋₆ alkyl optionally substituted with up to 5 fluoro, and C₁₋₆alkoxy optionally substituted with up to 5 fluoro;

R₂₀ is hydrogen or aryl optionally substituted with one or moresubstituents each independently selected from the group consisting ofhalo, cyano, nitro, hydroxy, C₁₋₆ alkyl optionally substituted with upto 5 fluoro, and C1-6 alkoxy optionally substituted with up to 5 fluoro;and

wherein when Y is NR₈R₉ then R₈ and R₂ optionally come together to beC₁₋₃ alkyl linking together as a ring,

with the proviso when R₈ comes together with R₂ to be C₁₋₃ alkyl linkingtogether as a ring then R₄, is not substituted with hydroxyl.

In one aspect, the CDC42 inhibitor may comprise Formula II, or apharmaceutically acceptable salt thereof

wherein:

Y is NR₈R₉,

R₈ is hydrogen; and R₉ is C₁₋₆ alkyl, said C₁₋₆ alkyl, optionallysubstituted with one or more substituents each independently selectedfrom the group consisting of hydroxy, R₁₉ or OR₂₀;

R₁₉ is phenyl optionally substituted with one or more substituents eachindependently selected from the group consisting of halo, cyano, C₁₋₆alkyl optionally substituted with up to 5 fluoro, and C₁₋₆ alkoxyoptionally substituted with up to 5 fluoro; and

R₂₀ is hydrogen or phenyl optionally substituted with one or moresubstituents each independently selected from the group consisting ofhalo, cyano, nitro, hydroxy, C₁₋₆ alkyl optionally substituted with upto 5 fluoro, and C₁₋₆ alkoxy optionally substituted with up to 5 fluoro.

In one aspect, the CDC42 inhibitor may comprise Formula IIa (CASIN), ora pharmaceutically acceptable salt thereof

In one aspect, the RHOA inhibitor may comprise (Formula III) (G04), or apharmaceutically acceptable salt thereof.

In one aspect, the composition may comprise the Formula 1b (NSC23766) ora pharmaceutically acceptable salt thereof, Formula IIa (CASIN) or apharmaceutically acceptable salt thereof; Formula III (G04) or apharmaceutically acceptable salt thereof, and combinations thereof.

Any amine, hydroxy, or carboxyl side chain on the compounds of the maybe esterified or amidified. The procedures and specific groups to beused to achieve this end are known to those of skill in the art.

Synthesis of one or more of the above-referenced compounds may bedescribes in, for example, U.S. Pat. No. 8,383,124 to Zheng, issued Feb.26, 2013; U.S. Pat. No. 7,612,080 to Zheng et al., issued Nov. 3, 2009;and U.S. Pat. No. 7,517,890 to Zheng et al, issued Apr. 14, 2009, thecontents of which are incorporated by reference for all purposes.

The active agent can form salts, which are also within the scope of thepreferred embodiments. Reference to a compound of the active agentherein is understood to include reference to salts thereof, unlessotherwise indicated. The term “salt(s)”, as employed herein, denotesacidic and/or basic salts formed with inorganic and/or organic acids andbases. In addition, when an active agent contains both a basic moiety,such as, but not limited to an amine or a pyridine or imidazole ring,and an acidic moiety, such as, but not limited to a carboxylic acid,zwitterions (“inner salts”) can be formed and are included within theterm “salt(s)” as used herein. Pharmaceutically acceptable (e.g.,non-toxic, physiologically acceptable) salts are preferred, althoughother salts are also useful, e.g., in isolation or purification steps,which can be employed during preparation. Salts of the compounds of theactive agent can be formed, for example, by reacting a compound of theactive agent with an amount of acid or base, such as an equivalentamount, in a medium such as one in which the salt precipitates or in anaqueous medium followed by lyophilization.

The active agents which contain a basic moiety, such as, but not limitedto an amine or a pyridine or imidazole ring, can form salts with avariety of organic and inorganic acids. Exemplary acid addition saltsinclude acetates (such as those formed with acetic acid or trihaloaceticacid, for example, trifluoroacetic acid), adipates, alginates,ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates,borates, butyrates, citrates, camphorates, camphorsulfonates,cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides (formed withhydrochloric acid), hydrobromides (formed with hydrogen bromide),hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed withmaleic acid), methanesulfonates (formed with methanesulfonic acid),2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates,persulfates, 3-phenylpropionates, phosphates, picrates, pivalates,propionates, salicylates, succinates, sulfates (such as those formedwith sulfuric acid), sulfonates (such as those mentioned herein),tartrates, thiocyanates, toluenesulfonates such as tosylates,undecanoates, and the like.

The active agents that contain an acidic moiety, such as, but notlimited to a carboxylic acid, may form salts with a variety of organicand inorganic bases. Exemplary basic salts include ammonium salts,alkali metal salts such as sodium, lithium, and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases (for example, organic amines) such as benzathines,dicyclohexylamines, hydrabamines [formed withN,N-bis(dehydro-abietyl)ethylenediamine], N-methyl-D-glutamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine and the like. Basic nitrogen-containing groups canbe quaternized with agents such as lower alkyl halides (e.g., methyl,ethyl, propyl, and butyl chlorides, bromides and iodides), dialkylsulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), longchain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides), aralkyl halides (e.g., benzyl and phenethylbromides), and others.

Active agent, and salts thereof, can exist in their tautomeric form (forexample, as an amide or imino ether). All such tautomeric forms arecontemplated herein as part of the preferred embodiments.

All stereoisomers of the present compounds, such as those, for example,which can exist due to asymmetric carbons on any of the substituents,including enantiomeric forms (which can exist even in the absence ofasymmetric carbons) and diastereomeric forms, are contemplated andwithin the scope of the preferred embodiments. Individual stereoisomersof the compounds of the preferred embodiments can, for example, besubstantially free of other isomers, or can be admixed, for example, asracemates or with all other or other selected, stereoisomers. The chiralcenters of the preferred embodiments can have the S or R configurationas defined by the IUPAC 1974 Recommendations.

When the compounds are in the forms of salts, they may comprisepharmaceutically acceptable salts. Such salts may includepharmaceutically acceptable acid addition salts, pharmaceuticallyacceptable base addition salts, pharmaceutically acceptable metal salts,ammonium and alkylated ammonium salts. Acid addition salts include saltsof inorganic acids as well as organic acids. Representative examples ofsuitable inorganic acids include hydrochloric, hydrobromic, hydroiodic,phosphoric, sulfuric, nitric acids and the like. Representative examplesof suitable organic acids include formic, acetic, trichloroacetic,trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic,citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic,glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates,phosphates, perchlorates, borates, acetates, benzoates,hydroxynaphthoates, glycerophosphates, ketoglutarates and the like.Examples of metal salts include lithium, sodium, potassium, magnesiumsalts and the like. Examples of ammonium and alkylated ammonium saltsinclude ammonium, methylammonium, dimethylammonium, trimethylammonium,ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium,tetramethylammonium salts and the like. Examples of organic basesinclude lysine, arginine, guanidine, diethanolamine, choline and thelike.

The pharmaceutically acceptable salts may be prepared by methods knownto one of ordinary skill in the art. For example, by reacting the activeagent with 1 to 4 equivalents of a base such as sodium hydroxide, sodiummethoxide, sodium hydride, potassium t-butoxide, calcium hydroxide,magnesium hydroxide and the like, in solvents like ether, THF, methanol,t-butanol, dioxane, isopropanol, ethanol, etc. Mixture of solvents canbe used. Organic bases like lysine, arginine, diethanolamine, choline,guandine and their derivatives etc. can also be used. Alternatively,acid addition salts wherever applicable are prepared by treatment withacids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid, phosphoric acid, p-toluenesulphonic acid, methanesulfonic acid,fonic acid, acetic acid, citric acid, maleic acid salicylic acid,hydroxynaphthoic acid, ascorbic acid, palmitic acid, succinic acid,benzoic acid, benzenesulfonic acid, tartaric acid and the like insolvents like ethyl acetate, ether, alcohols, acetone, THF, dioxane,etc. Mixture of solvents can also be used.

In one aspect, any of the above-described compositions may comprise aphysiologically acceptable carrier. In one aspect, the physiologicallyacceptable carrier may comprise a buffer. In one aspect, thephysiologically acceptable carrier may be selected from saline,phosphate buffered saline, Tris buffered saline, Hank's buffered saline,water, or a combination thereof. In one aspect, the physiologicallyacceptable carrier may comprise an electrolyte solution.

In one aspect, the RAC inhibitor, the CDC42 inhibitor, and the RHOAinhibitor may be present in a carrier at a concentration, in combinationor separately, of at least about 10 μM, or at least about 25 μM, atleast about 50 μM, at least about 100 μM, at least about 200 μM, atleast about 500 μM, at least about 1 mM, at least about 10 mM, at leastabout 50 mM, at least about 100 mM, or at least about 1 M.

In one aspect, the RAC inhibitor may be present at a concentration offrom about 10 μM to about 500 μM, or from about 25 μM to about 400 μM,or from about 50 μM to about 300 μM, or from about 75 μM to about 200μM, or from about 100 μM to about 150 μM, or about 50 μM.

In one aspect, the CDC42 inhibitor may be present at a concentration offrom about 5 μM to about 500 μM, or from about 10 μM to about 400 μM, orfrom about 25 μM to about 300 μM, or from about 50 μM to about 200 μM,or from about 75 μM to about 150 μM, or about 10 μM.

In one aspect the RHOA inhibitor may be present at a concentration offrom about 10 μM to about 500 μM, or from about 25 μM to about 400 μM,or from about 50 μM to about 300 μM, or from about 75 μM to about 200μM, or from about 100 μM to about 150 μM, or about 75 μM.

In further aspects, the described compositions may comprise an additiveselected from NaCl, KCl, CaCl2, MgCl₂, MgSO₄, Na³ citrate, citric acid,NaHCO₃, Na phosphate, Na acetate, Na gluconate, glucose, maltose,mannitol, and combinations thereof. The described compositions maycomprise an additive selected from NaCl, KCl, CaCl₂, MgCl₂, MgSO₄, Na³citrate, citric acid, NaHCO₃, Na phosphate, Na acetate, Na gluconate,glucose, maltose, mannitol, and combinations thereof, wherein saidadditive may be present in an amount of from about 0.5 mmol/L to about150 mmol/L.

In further aspects, the described compositions may comprise one or moreingredients selected from D-ribose, D-glucose, Hanks solution, Hepessolution, bovine serum albumin, tic anticoagulant peptide and sterilewater, or combinations thereof.

In one aspect, the composition may comprise an auxiliary substanceselected from pH adjusting and buffering agents, tonicity adjustingagents, stabilizers, wetting agents, and combinations thereof.

In one aspect, the composition may have a pH of from about 5 to about 8,or from about 6 to about 7, or about 6.8 to about 7.4.

In one aspect, the composition may be isotonic.

In yet further aspects, the composition may comprise an additionaltherapeutic agent.

A method for storing platelets is also described. In this aspect, themethod comprises the step of storing platelets in a composition asdisclosed herein.

In one aspect, the method may comprise contacting platelets with asolution comprising an RAC inhibitor, an CDC42 inhibitor, and/or a RHOAinhibitor, wherein the one or more actives are present in a carrier at aconcentration, in combination or separately, of at least about 10 μM, orat least about 25 μM, at least about 50 μM, at least about 100 μM, atleast about 200 μM, at least about 500 μM, at least about 1 mM, at leastabout 10 mM, at least about 50 mM, at least about 100 mM, or at leastabout 1 M.

In one aspect, the method may comprise contacting platelets with asolution comprising an RAC inhibitor present in a solution at aconcentration of from about 10 μM to about 500 μM, or from about 25 μMto about 400 μM, or from about 50 μM to about 300 μM, or from about 75μM to about 200 μM, or from about 100 μM to about 150 μM, or about 50μM.

In one aspect, the method may comprise contacting platelets with asolution comprising a CDC42 inhibitor present in a solution at aconcentration of from about 5 μM to about 500 μM, or from about 10 μM toabout 400 μM, or from about 25 μM to about 300 μM, or from about 50 μMto about 200 μM, or from about 75 μM to about 150 μM, or about 10 μM.

In one aspect, the method may comprise contacting platelets with asolution comprising an RHOA inhibitor present in a solution at aconcentration of from about 10 μM to about 500 μM, or from about 25 μMto about 400 μM, or from about 50 μM to about 300 μM, or from about 75μM to about 200 μM, or from about 100 μM to about 150 μM, or about 75μM.

In one aspect, the storage step may be carried out at a temperature offrom about 1° C. to about 20° C., or at about 1° C., or about 2° C., orabout 3° C., or about 4° C., or about 5° C., or about 6° C., or about 7°C., or about 8° C., or about 9° C., or about 10° C., or about 11° C., orabout 12° C., or about 13° C., or about 14° C., or about 15° C., orabout 16° C.

In one aspect, the storage step may be carried out for a period of timeof from about 7 to 20 days, or from about 10 to 15 days, or greater than7 days, or greater than 8 days, or greater than 9 days, or greater than10 days, or greater than 11 days, or greater than 12 days, or greaterthan 13 days, or greater than two weeks.

In one aspect, the composition may be used in an amount sufficient toinhibit a platelet damaging activity selected from polymerization ofF-actin, depolymerization of F-actin, actomyosin contraction, tubulinpolymerization, spectrin anchorage, or combinations thereof.

In one aspect, applying the described methods, the platelet survival maybe greater than about 65%, or greater than about 70%, or greater thanabout 75%, or greater than about 80%, or greater than about 85%, orgreater than about 90%, or greater than about 95%, or greater than about99% after a storage period of 24 hours at 5 C.

In one aspect, a method of reversing platelet activation is alsodisclosed, comprising contacting activated platelets with a compositionas described herein. In one aspect, the contacting step may be carriedout at a temperature of about 0° C. In a yet further aspect, thedisclosed compositions may be used to reverse refrigeration storagelesions in platelets. In this aspect, platelets having refrigerationstorage lesions may be contacted with a composition as disclosed herein,for a period of time sufficient to reverse refrigeration storagelesions.

EXAMPLES

Applicant has tested the ability of three chemical inhibitors, CASIN,NSC23766 and G04, targeting Cdc42, Rac1 and RhoA, respectively (FIG. 2),to inhibit the activity of the GTPases and the consequentcytoskeleton-dependent functions of human platelets. A rationallydeveloped inhibitor of Cdc42 with activity on activation by guaninenucleotide exchange factors is CASIN (29, Nature Biotech underrevision). CASIN was recently discovered by Zheng group as a smallmolecule that recognizes a pocket domain that specifically blocks theability of GEF interaction with Cdc42 (FIG. 3A). CASIN is found tosuppress of hematopoietic stem cell aging through its specific Cdc42inhibitory activity (29). In platelets, it inhibits Cdc42 activation(FIG. 3B) and prevents collagen-induced platelet shape changes (FIG.3C). These changes depend on integrin signaling and involve F-actinpolymerization and filopodia formation as demonstrated by phalloidingstaining (FIG. 3C, lower panels). Cdc42 inhibition by CASIN results inprevention of collagen-induced platelet aggregation (FIG. 3D) that canbe reversed by a washout of the inhibitor (FIG. 3D, upper and lowerpanels).

Similarly, Applicant has identified an inhibitor of Rac, NSC23766(30-34), with an ability to impair Rac1 activation by multipleactivating signals which may be crucial in platelet signaling associatedwith GpIb/GpX (35). NSC23766 was discovered in a structure-based virtualscreening of compounds that fit into a surface groove of Rac1 known tobe critical for guanine nucleotide exchange factor specification (34)(FIG. 4A). NSC23766 can effectively block Rac1 activation (FIG. 4B) andRac-dependent cytoskeletal rearrangements (actin lamellopodia) ofplatelets stimulated by collagen (30, 32, 35), indicating its abilityfor suppressing collagen-integrin dependent signaling (FIG. 4C).Finally, NSC23766 reversibly inhibits, in a dose-dependent fashion,platelet aggregation induced by collagen (FIG. 4D, upper and lowerpanels).

Finally, G04/Rhosin was also developed by us as a RhoA GTPase activationsite inhibitor that transiently and specifically blocks RhoA activityand RhoA-mediated signaling functions (36, 37). G04 contains twoaromatic rings tethered by a linker, and it binds to the surface areasandwiching Trp58 of RhoA (FIG. 5A) with a submicromolar Kd andeffectively inhibits GEF-catalyzed RhoA activation. In platelets, G04specifically inhibits collagen-induced RhoA activity (FIG. 5B) andRhoA-mediated cellular functions including fibrinogen-dependent plateletspreading (FIG. 5C) and collagen-dependent platelet aggregation (FIG.5D). Effect by collagen or thrombin is reversible (FIG. 5D, lower).

In addition to the reversibility, each inhibitor transiently mimics theeffects of Cdc42, Rac1, or RhoA gene knockout in platelets,respectively, and does not show any additive effects in the respectiveknockout cells (data not shown), indicating their specificity and a lackof toxicity.

A combination of CASIN (10 μM), G04 (75 μM) and/or NSC23766 were used inC57Bl/6 murine carboxyfluorescein-lateled platelets incubated at 0° C.or 5° C. for 3.5 hours. After re-warming, platelets were infused in 5congenic mice (per group) and compared with control (room temperaturestored) and refrigerated, untreated platelet group (FIG. 7). While thecombination of NSC23766, G04 and CASIN result in close-to-completereversal of the survival deficiency induced by refrigerated storage,NSC23766 alone or NSC23766 in combination with CASIN or G04 did notresult in significant reversal of the storage lesion of platelets (FIG.7). In a second experiment focused on the specific combinations withmore activity to reverse the survival impairment associated with the useof Rho GTPase inhibitors, we analyzed the 24-hour recovery (%) andsurvival (hours) of platelets that had been stored in presence ofseveral combinations of drugs (FIG. 6A).

A triple combination of CASIN, G04 and NSC23766 resulted in asignificant improved recovery (FIG. 6B) and survival (FIG. 6C) ofrefrigerated platelets after infusion. Use of the same triple inhibitorcombination during storage of refrigerated platelets at 50 C instead of0° C. resulted in complete reversal of the recovery and survival ofrefrigerated platelets in vivo (FIG. 6D) as determined by multi-hitregression analysis (COSTCO software) and ANOVA test with Bonferronicorrection for statistical differences. Applicant found that the 24-hourrecovery of platelets refrigerated was significantly reduced (˜20%)compared with the group control (stored at room temperature, p<0.01).Interestingly, the shortened recovery was completely reversed byincubation with the cocktail of three inhibitors (FIG. 6D). Altogether,these data indicate that the inhibitory combination of Cdc42, Rac1, andRhoA with CASIN, NSC23766 and G04 can specifically and reversiblyinhibit platelet activation and may be useful in a reversal ofrefrigeration storage lesion in platelets.

Finally, Rho family GTPase inhibitors can prevent human platelet storagelesion in vitro as assessed by microparticle formation and content ofGpIb (CD42b) in microparticles. Applicant found that the use ofinhibitors resulted in reversal of the refrigeration dependentmicroparticle generation (FIGS. 8A-B). Finally, Applicant analyzed theformation of lipid raft microdomains on the platelet membrane bydifferential analysis in lysates enriched in Triton-X-100 resistantmembrane fragments in human platelets stored refrigerated for 3 and 7days (FIG. 9). We found a complete correlation between the results ofsurvival of refrigerated platelets in presence of inhibitor combinationswith the formation of lipid raft microdomains, suggesting that theinhibitor combination containing G04, NSC23766 and CASIN results inreversal of refrigeration-induced lipid raft formation. Interestingly,washing out G04, NSC23766 and CASIN results in restoration of the samelevels of lipid rafts as found in 3 or 7 day stored platelets at roomtemperature (FIG. 9), suggesting that the removal of these inhibitorsresults in platelet membrane rheological modifications similar to theones observed in unprocessed, standard storage platelets for the sameperiod of time (FIG. 9). These data provide proof-of-concept that thelong-term storage (6 days) of platelets in plasma containing Rho GTPaseinhibitors is not deleterious of platelets but can further prevent theirstorage lesion-associated activation.

Thus, Applicant's data strongly support that reversible inhibition ofmultiple Rho family of GTPases by chemical inhibitors can significantlyprevent refrigerated storage damage and improve platelet survival andtransfusion function after refrigeration by interference with lipid raftmicrodomain formation, actomyosin dynamics and GPIb clustering inmembrane microdomains.

All percentages and ratios are calculated by weight unless otherwiseindicated.

All percentages and ratios are calculated based on the total compositionunless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method for preserving platelet function and/oractivity in a platelet subjected to cold storage comprising contactingsaid platelet with a composition comprising a RHOA inhibitor selectedfrom:

or pharmaceutically acceptable salt thereof,

or pharmaceutically acceptable salt thereof, or combinations thereof. 2.The method of claim 1 wherein said RHOA inhibitor is R-G04.
 3. Themethod of claim 1 wherein said RHOA inhibitor is S-G04.
 4. The method ofclaim 1 wherein said RHOA inhibitor is present at a concentration insaid composition in an amount of at least about 10 μM.
 5. The method ofclaim 1, wherein said RHOA inhibitor is present at a concentration insaid composition in an amount of from about 10 μM to about 500 μM. 6.The method of claim 1 wherein said composition comprises a buffer. 7.The method of claim 1 wherein said composition comprises a carrierselected from saline, phosphate buffered saline, Tris buffered saline,Hank's buffered saline, an electrolyte solution, water, or a combinationthereof.
 8. The method of claim 1 wherein said composition comprises anadditive selected from NaCl, KCl, CaCl₂, MgCl₂, MgSO₄, Na₃ citrate,citric acid, NaHCO₃, Na phosphate, Na acetate, Na gluconate, glucose,maltose, mannitol, D-ribose, D-glucose, Hanks solution, Hepes solution,bovine serum albumin, tic anticoagulant peptide and sterile water, a pHadjusting agent, a pH buffering agent, a tonicity adjusting agent, astabilizer, a wetting agent, and combinations thereof.
 9. The method ofclaim 8, wherein said additive is present in an amount of from about 0.5mmol/L to about 150 mmol/L.
 10. The method of claim 1 wherein saidcomposition has a pH of from about 5 to about
 8. 11. The method of claim1 wherein said composition is isotonic.
 12. The method of claim 1wherein said composition comprises a therapeutic agent.
 13. The methodof claim 1, wherein said cold storage is carried out at a temperature offrom about 0° C. to about 20° C.
 14. The method of claim 1, wherein saidcold storage is carried out for a period of time of from about 7 toabout 20 days.
 15. The method of claim 1 wherein said RHOA inhibitor ispresent in an amount sufficient to inhibit a platelet damaging activityselected from polymerization of F-actin, depolymerization of F-actin,actomyosin contraction, tubulin polymerization, and spectrin anchorage.16. The method of claim 1 wherein platelet survival is greater thanabout 65% after a storage period of 24 hours at 5° C.
 17. A method ofreversing refrigeration storage lesion in platelets comprisingcontacting platelets having refrigeration storage lesions with thecomposition of claim
 1. 18. A method for preserving platelet functionand/or activity in a platelet subjected to cold storage comprisingcontacting said platelet with a composition consisting essentially of aRHOA inhibitor selected from:

or pharmaceutically acceptable salt thereof,

or pharmaceutically acceptable salt thereof, or combinations thereof.19. A method of reversing refrigeration storage lesion in plateletscomprising contacting platelets having refrigeration storage lesionswith the composition of claim 18.